Decreased insulin action, impaired insulin secretion and increased adiposity are important risk factors for development of type 2 diabetes. These risk factors are present even when individuals have both normal fasting and two hour glucose concentrations indicating a very early role for decreased insulin secretion in the development of type 2 diabetes. In previous work we had found several bimodally expressed genes in muscle tissue. One of these, HLA-DRB1, was associated with lower risk for diabetes and higher insulin secretion in Pima Indians. As the HLA system is involve in immune regulation, therefore presence of protective HLA haplotype indicates involvement of autoimmunity in progression to type 2 diabetes in Pima Indians. To further explore this, we performed a pilot study in individuals with and without the HLA-DRB1 locus who were also discordant for diabetes status, examining both immunoprofiling of over 9,000 potential proteins and T-cell receptor genes. Our preliminary results demonstrate 200 possible antibodies that might be markers of progression to type 2 diabetes, and differences in frequencies of T-cell receptor genes between those with and without diabetes. We are planning follow-up studies in which we will attempt to validate a selection of the antibodies and further investigate the association of these T-cell receptor gene frequencies with measurements of insulin action and insulin secretion. We are continuing to investigate additional factors which control insulin secretion and insulin action. Individuals who are undergoing bariatric surgery using the following techniques: Roux-en-Y gastric bypass (RYGBP), laporoscopic band, or gastric sleeve will undergo measures of insulin action, insulin secretion and meal tests prior to and one month following the surgery. Preliminary results indicate that in individuals without diabetes one month of after surgery those who underwent RYGBP had dramatic improvements in hepatic insulin sensitivity, and more rapid glucose and hence insulin responses during meal tests. This explains the greater improvement in glycemia seen in those with diabetes undergoing RYGBP compared with other the laporoscopic band. We are continuing to perform more detailed measurements of insulin action and secretion and are continuing longer term follow-up to determine the durability of these shorter term changes. We are also comparing individuals undergoing sleeves to RYGBP. Previous predictors of weight gain based on this study have included, higher respiratory quotient, higher insulin mediated glucose uptake, lower free T3, and relatively lower energy expenditure. As variability in energy expenditure remains a potential mediator of weight change we have continued to evaluate factors related to metabolic rate.. We had also found that the concentration of the endocannabinoid , 2-arachidonoylglycerol (2-AG), in cerebrospinal fluid is higher in American Indians, and that CSF oleoylethanolamide was associated with energy expenditure. Endocannabinoids are fatty acids derivatives. In rodent models long chain fatty acids have specific roles in regulating food intake and hepatic glucose output. We measured long chain saturated, mono-unsaturated and poly-unsaturated fatty acids in CSF and investigated their associations with metabolic measurements. We found that specific mono-unsaturated fatty acids (18:1 and 16:1 or oleic and palmitoleic acid respectively) were associated with lower respiratory quotient (a measure of the ratio of carbohydrate to lipid oxidation) and lower glucose concentrations during an oral glucose tolerance test. Furthermore, we found that very long chain fatty acid concentrations in the CSF were inversely associated with 24 hour energy expenditure. These results indicate specific roles for long chain fatty acids as central signaling molecules with important peripheral effects. We are continuing to examine the role of these specific long chain fatty acids centrally and in the periphery and their effects on insulin action, energy expenditure and food intake. Genetic factors underlie adiposity and its risk factors. Mutations in the melanocortin 4 receptor gene are associated with increased body mass index and lower 24 hour energy expenditure in humans. We investigated the lifecourse effect of the MC4R mutation and found that the accelerated weight gain occurred in childhood but not in adulthood indicating a more potent effect of this mutation in early life. Furthermore, we found that presence of MC4R mutations predicted development of diabetes in childhood independent of body weight. We are now investigating how MC4R mutations may affect downstream mediators that lead to accelerated weight gain.